Functionalized gold nanoparticles (AuNPs) offer a promising solution to the challenge of targeted drug delivery, and they possess unique spectroscopic properties that facilitate optical detection and diagnostics. Several AuNP- based systems have been designed to treat cancer and neurodegenerative diseases, and treatments are envisioned for infectious disease as well. Protein molecules spontaneously adsorb to AuNP surfaces, and one approach to designing functionalized AuNPs is to bind active enzymes directly using this spontaneous process. Unfortunately, not all enzymes remain active on the AuNP surface, and it is currently impossible to predict whether an enzyme will retain its activity when adsorbed. This represents a major hurdle for the field, and as a result general-purpose, protein-functionalized AuNPs remain a distant goal. Before the long-term goal of creating new nanoparticle-based therapies is realized, however, an improved understanding of protein structure on the AuNP surface must be developed. This improved understanding is the immediate objective of this proposal. Preliminary data is presented demonstrating the feasibility of using NMR spectroscopy to investigate the structure of adsorbed proteins. NMR has the potential to reveal structural detail at the resolution of individual residues, bet recent advances in large-molecule NMR have not been adequately leveraged in this field. This work applies innovative NMR methodologies to this challenging and important problem. Specifically, this proposal has three specific aims: (1) Identify the driving residues behind AuNP-protein interactions. The approach for this aim is to use newly-developed pulse programs to study the initial stages of protein association. (2) Determine the structure and orientation of AuNP surface proteins. In this aim, hydrogen/deuterium exchange methods will be combined with side-chain isotopic labeling to yield a picture of structure on the AuNP surface. (3) Synthesize functionalized AuNPs with carbonic anhydrase activity. This final aim will develop a general model system for reliably functionalizing AuNPs with enzymatic activity. Through these aims, a much better understanding of protein structure on AuNPs will be attained, and general design principles for creating functionalized AuNPs will be developed. Using the NMR approaches employed here, it will be possible to examine structural deformations of adsorbed proteins, and the behavior of proteins during the initial stages of binding will be better understood. These discoveries will have a positive impact because they will enable researchers to develop AuNP-based therapeutics and diagnostics much more quickly and efficiently, and this will lead to markedly better nanotechnology-based tools in the biomedical sciences.